This project is devoted to examining the biochemical mechanisms responsible for the prethrombotic state in humans and developing new techniques for its detection. The plasma proteins involved in the clotting of blood are normally present in an inactive or minimally active form. These components include zymogens, cofactors and inhibitors. During the activation of the hemostatic mechanism each of these components is converted to a highly potent species. Since these molecular transitions are crucial for defining the hypercoagulable state, we have developed a number of sensitive and specific assays for detecting these events. The early phases of this grant will be devoted to validating these procedures as well as developing additional ancillary assay techniques of similar sensitivity and specificity. This methodology will then be utilized to examine the normal molecular processes which control the generation of thrombin and modulate its inhibition under in vivo conditions within the blood. We expect to determine the underlying reasons for the great differences that we have observed in the kinetic behavior of the prothrombin conversion mechanism within purified systems vis a vis complex physiologic fluids. In addition, we hope to show that the coagulation system is tonically active in normal individuals. This finding would have great conceptual importance for understanding the physiology of the hemostatic mechanism. It would demonstrate that thrombin is continually evolved in the vascular system of humans, albeit at levels far below those required for fibrin deposition and within the range of normal inhibitory mechanism. Furthermore, we hope to prove that an elevation in the normal levels of thrombin production correlates with the eventual onset of thrombotic disease. Thus, our investigations may provide an approach for identifying individuals with hyperactive coagulation systems who are at risk for these disorders. Growing experience in these areas may permit the design of prophylactic measures for preventing these catastrophic events.